S5 Fig: Analysis of MCPyV ALTO transcriptional regulation. ![]() The P value was determined by unpaired, two-tailed t-test, *<0.05, **<0.01. Error bars represent the SD (n = 3 biological replicates). (G) qRT-PCR of circALTO of 293T co-transfected with control or METT元/14 siRNAs and circALTO1/2 construct. (F) Western blots for METT元 and Flag from 293T co-transfected with control or METT元/14 siRNA and circALTO1 construct. After 48 h of transfection, the cells were fixed and stained for FLAG (green), and DAPI (blue). (E) 293T cells were transfected with the Flag-circALTO1/2 plasmids alone and Flag-circALTO1/2 with indicated siRNAs. (D) Western blot for FLAG from 293 T cells co-transfected with Flag-circALTO2. ![]() (C) qRT–PCR analysis for circALTO and ALTO mRNA in co-transfected Flag-circALTO2 constructs 293 T cells with siRNAs (see S3A Fig). (B) Western blot for FLAG from 293T cells co-transfected with Flag-circALTO1 and the indicated siRNA. (A) qRT-PCR analysis for circALTO and ALTO mRNA in co-transfected Flag-circALTO1 constructs 293T cells treated with the indicated siRNAs (see S3A Fig). S4 Fig: Characterization of circALTOs with siRNAs. (D) Under conditions where expression of circALTO1 enhanced reporter gene expression, it does not alleviate MCPyV-miRNA-mediated repression. Under conditions where Rluc is being targeted by MCPyV miRNA, this reporter and the non-miRNA-targeted FF-Luc both display enhanced expression in the presence of circALTO. (C) circALTO1 enhanced reporter gene expression irrespective of MCPyV miRNA activity. (B) 293 cells were transfected with Renilla luciferase reporter with MCPyV miRNA (MCV339) and the indicated plasmids. Firefly luciferase served as a transfection control and Renilla luciferase levels are plotted normalized relative to firefly luciferase levels (n = 2 biological replicates). (A) 293T cells were transfected with Renilla luciferase reporter with MCPyV miRNA (MCV350) and the indicated plasmids including pcDNA3.1 control vector, pCDNA circALTO1 or pCDNA circALTO2 expression plasmid. S3 Fig: CircALTO functions as a transcriptional inducer but not a miRNA sponge in vitro. Error bars represent the SD of three biological replicates. Values normalized to the enriched fraction. MALAT1 and ACTB served as fractionation controls. (E) Nuclear and cytoplasmic fractionation assay of 293 T cells co-transfected with circALTO1 construct performed by qRT-PCR. Arrows indicates RNase R resistant band circALTO2. (D) Northern blot of total RNA from 293T cells co-transfected with vector control and pcDNA3.1-circALTO2 constructs probed with linear ALTO after RNase R treatment. Primers designed from CMV promoter region to the regions that flanked circALTOs. (C) Readthrough transcript from 293T cells co-transfected with pcDNA3.1-circALTO1/2 constructs with or without RNase R treatment were analyzed by RT-PCR. Sanger sequencing of PCR products showed the non-specific backsplice junction with the insertion of additional nucleotides (Right panel). (B) The formation of circALTOs from 293T cells co-transfected with pcDNA3.1-circALTO1/2 constructs were confirmed by RT-PCR (Left panel). The location of QKI sites, ORF, 3xFLAG epitope-tag (present in ‘FLAG’), mutated splice sites (short for ‘SASD’), and siRNAs used in subsequent knockdown assay were indicated in the diagram. (A) Schematic diagram of circALTO1 (Left panel) and circALTO2 (Right panel) expression constructs generated in vitro. S2 Fig: Characterization of circALTO constructs. ![]() Wang, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing 1, 10, * Sullivan, Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Writing – review & editing, 3 and Richard C. Feltkamp, Conceptualization, Investigation, Resources, Writing – review & editing, 9 Christopher S. Buck, Conceptualization, Resources, Writing – review & editing, 8 Mariet C. Smith, Resources, 6 Leslie Rosen, Resources, 6 Louisa Verlinden, Resources, 7 Denise A. Scherer, Methodology, 4 Clay Cockerell, Resources, 1 Taylor R. Choi, Conceptualization, Investigation, 3 Elysha Kolitz, Investigation, Writing – review & editing, 1 Yating Chen, Investigation, 3 Clair Crewe, Investigation, Methodology, 4 Nicholas J. Lee, Investigation, Methodology, 1 Jiwoong Kim, Data curation, Formal analysis, Methodology, Software, Writing – review & editing, 2 Joon H. Rong Yang, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing, 1 Eunice E.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |